Friedreich's Ataxia (FRDA) affects >20.000 individuals in Caucasian populations. Generally within 10 to 15 from onset it leads to loss of deambulation and complete disability, with premature death often caused by cardiac insufficiency1. Symptoms usually appear late in the first decade or early in the second decade of life, and include gait instability and general clumsiness. Skeletal abnormalities, such as scoliosis or pes cavus, may be already present. Gait ataxia has both cerebellar and sensory features, involves truncus and limbs, and is progressive and generally unremitting. Swaying is common and, as it becomes more severe, eventually requires constant support and wheelchair use. Dysarthria occurs early in the disease and progress to complete speech impairment. Dysphagia is a late feature and may require artificial feeding. Ventricular hypertrophy characterizes the cardiac picture, and may progressively lead to congestive heart failure and fatal arrhythmias. A significant minority of patients also develop diabetes mellitus, by not yet clearly defined mechanisms2.
FRDA is caused by homozygous hyperexpansion of GAA triplets within the first intron of the FXN gene, an highly conserved five-exon gene located on the long arm of human chromosome 9, coding for the protein frataxin. Pathological GAA expansions (from ˜70 to >1,000 triplets) result in “sticky” DNA structures and epigenetic changes that severely reduce transcription of the FXN gene. FRDA patients live with 10-30% residual frataxin, the severity of the disease being directly proportional to the number of GAA triplets and to the consequent degree of frataxin reduction. A minority of FRDA patients, so called compound heterozygotes, has pathological GAA expansions on one FXN allele and loss-of-function mutations on the other. Complete loss of frataxin is not compatible with life, in all higher species examined3.
Human frataxin is synthesized as a 210 amino acid (aa) precursor that is rapidly targeted to the mitochondria. Upon entrance into the mitochondria, the frataxin precursor undergoes a two-step proteolytic processing, mediated by the mitochondrial protein peptidase (MPP). The resulting mature frataxin is a 130aa globular polypeptide that mostly resides within the mitochondrial matrix4,5, but that can be also found outside the mitochondria6,7, where it might interact with and regulate cytosolic aconitase/IRP18. Frataxin may bind iron directly and act either as an iron donor9,10 or as an iron sensor involved in the proper functioning of the iron-sulphur cluster (ISC) machinery11. Frataxin-defective cells have reduced activity of ISC-containing enzymes, a general imbalance in intracellular iron distribution and increased sensitivity to oxidative stress.
There is currently no specific therapy to prevent the progression of the disease12. Most therapeutic approaches are aimed at reducing mitochondrial dysfunction and are based on the use of anti-oxidant or iron chelators13,14. Beside this, as levels of residual frataxin are crucial in determining the severity of the disease, many efforts have been put in the identification of molecules that increase frataxin transcription15,16. However, no studies have been so far reported regarding neither the physiological turnover of this protein in humans, nor any factors that can modulate its stability. Therefore, the comprehension of the molecular mechanisms that regulate frataxin protein stability might provide fundamental information towards the design of new therapeutic approaches.
Although the maturation process of frataxin has been well characterized, no information is available concerning the biology of frataxin degradation. Since the Ubiquitin-Proteasome System (UPS) is the major pathway for regulated intracellular protein degradation in higher eukaryotes, this pathway was investigated for its involvement in the control of frataxin turnover17.